PROJECT SUMMARY Approximately 8,000 people in the U.S. are diagnosed with germ cell tumors (GCTs) each year, and the vast majority are young men who develop testicular GCTs. Most patients are cured with conventional chemotherapy, although 30% recur, and half of such patients ultimately succumb to their disease. Given the long life expectancy of these patients, when death from GCT occurs, it accounts for among the greatest number of life years lost of any non-childhood malignancy representing. Our previous studies have demonstrated that GCTs exhibit an extreme burden of reciprocal loss of heterozygosity (RLOH) and high degree of mitochondrial priming for apoptosis. The goal of this proposal is to dissect the molecular features that initiate RLOH in GCTs, determine the relationship between RLOH and defect DNA checkpoints as tumors progress, and evaluate the ability of functional assays to identify highest risk disease prior to chemotherapy initiation. The long-term objective is to enable new mechanisms of patient stratification and identify new therapeutic targets for chemoresistant GCTs, currently an area of unmet medical need with extremely limited therapeutic options under investigation. This proposal is unique in that it leverages the extensive and novel resources at both the Dana-Farber Cancer Institute/Harvard Cancer Center and the Broad Institute of MIT and Harvard, along with an international team of collaborators, to overcome limited preclinical models of this disease and incorporate patient-centered assays focused on human tumor samples to address the hypotheses outlined herein. The proposed specific aims are: 1) To define the genetic defects associated with reciprocal loss of heterozygosity in primary germ cell tumors, 2) To identify the molecular features of tumor evolution leading to chemoresistant germ cell tumors, and 3) To assess the clinical utility of pluripotency markers as prognostic for GCT outcomes. These studies will define the meiotic defects underlying RLOH in GCTs, identify the secondary molecular defects that initiate lethal chemoresistance, and reveal targets for enhanced patient stratification and therapeutic development. In addition, these efforts will accelerate development of new computational algorithms that explore integrative molecular analyses of both the genome and epigenome to address specific hypotheses regarding oncogenic development and progression to chemoresistance that may have broad applicability. Finally, this project will accelerate the clinical and molecular characterization of GCTs, explore the underlying biology driving this rare tumor type, and serve more broadly as an innovative model for studying rare cancers.